Identifying the Metabolic Signatures of PPARD-Overexpressing Gastric Tumors.

Peroxisome proliferator-activated receptor delta (PPARD) is a nuclear receptor known to play an essential role in regulation of cell metabolism, cell proliferation, inflammation, and tumorigenesis in normal and cancer cells. Recently, we found that a newly generated villin-PPARD mouse model, in which PPARD is overexpressed in villin-positive gastric progenitor cells, demonstrated spontaneous development of large, invasive gastric tumors as the mice aged. However, the role of PPARD in regulation of downstream metabolism in normal gastric and tumor cells is elusive. The aim of the present study was to find PPARD-regulated downstream metabolic changes and to determine the potential significance of those changes to gastric tumorigenesis in mice. Hyperpolarized [1-13C] pyruvate magnetic resonance spectroscopy, nuclear magnetic resonance spectroscopy, and liquid chromatography-mass spectrometry were employed for metabolic profiling to determine the PPARD-regulated metabolite changes in PPARD mice at different ages during the development of gastric cancer, and the changes were compared to corresponding wild-type mice. Nuclear magnetic resonance spectroscopy-based metabolomic screening results showed higher levels of inosine monophosphate (p = 0.0054), uracil (p = 0.0205), phenylalanine (p = 0.017), glycine (p = 0.014), and isocitrate (p = 0.029) and lower levels of inosine (p = 0.0188) in 55-week-old PPARD mice than in 55-week-old wild-type mice. As the PPARD mice aged from 10 weeks to 35 weeks and 55 weeks, we observed significant changes in levels of the metabolites inosine monophosphate (p = 0.0054), adenosine monophosphate (p = 0.009), UDP-glucose (p = 0.0006), and oxypurinol (p = 0.039). Hyperpolarized [1-13C] pyruvate magnetic resonance spectroscopy performed to measure lactate flux in live 10-week-old PPARD mice with no gastric tumors and 35-week-old PPARD mice with gastric tumors did not reveal a significant difference in the ratio of lactate to total pyruvate plus lactate, indicating that this PPARD-induced spontaneous gastric tumor development does not require glycolysis as the main source of fuel for tumorigenesis. Liquid chromatography-mass spectrometry-based measurement of fatty acid levels showed lower linoleic acid, palmitic acid, oleic acid, and steric acid levels in 55-week-old PPARD mice than in 10-week-old PPARD mice, supporting fatty acid oxidation as a bioenergy source for PPARD-expressing gastric tumors.

Sugar nucleotide quantification by liquid chromatography tandem mass spectrometry reveals a distinct profile in Plasmodium falciparum sexual stage parasites.

The obligate intracellular lifestyle of Plasmodium falciparum and the difficulties in obtaining sufficient amounts of biological material have hampered the study of specific metabolic pathways in the malaria parasite. Thus, for example, the pools of sugar nucleotides required to fuel glycosylation reactions have never been studied in-depth in well-synchronized asexual parasites or in other stages of its life cycle. These metabolites are of critical importance, especially considering the renewed interest in the presence of N-, O-, and other glycans in key parasite proteins. In this work, we adapted a liquid chromatography tandem mass spectrometry (LC-MS/MS) method based on the use of porous graphitic carbon (PGC) columns and MS-friendly solvents to quantify sugar nucleotides in the malaria parasite. We report the thorough quantification of the pools of these metabolites throughout the intraerythrocytic cycle of P. falciparum The sensitivity of the method enabled, for the first time, the targeted analysis of these glycosylation precursors in gametocytes, the parasite sexual stages that are transmissible to the mosquito vector.

Integrated process design for biocatalytic synthesis by a Leloir Glycosyltransferase: UDP-glucose production with sucrose synthase.

Nucleotide sugar-dependent ("Leloir") glycosyltransferases (GTs), represent a new paradigm for the application of biocatalytic glycosylations to the production of fine chemicals. However, it remains to be shown that GT processes meet the high efficiency targets of industrial biotransformations. We demonstrate in this study of uridine-5'-diphosphate glucose (UDP-glc) production by sucrose synthase (from Acidithiobacillus caldus) that a holistic process design, involving coordinated development of biocatalyst production, biotransformation, and downstream processing (DSP) was vital for target achievement at ∼100 g scale synthesis. Constitutive expression in Escherichia coli shifted the recombinant protein production mainly to the stationary phase and enhanced the specific enzyme activity to a level (∼480 U/gcell dry weight ) suitable for whole-cell biotransformation. The UDP-glc production had excellent performance metrics of ∼100 gproduct /L, 86% yield (based on UDP), and a total turnover number of 103 gUDP-glc /gcell dry weight at a space-time yield of 10 g/L/h. Using efficient chromatography-free DSP, the UDP-glc was isolated in a single batch with ≥90% purity and in 73% isolated yield. Overall, the process would allow production of ∼0.7 kg of isolated product/L E. coli bioreactor culture, thus demonstrating how integrated process design promotes the practical use of a GT conversion. Biotechnol. Bioeng. 2017;114: 924-928. © 2016 Wiley Periodicals, Inc.

Hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry for the quantification of uridine diphosphate-glucose, uridine diphosphate-glucuronic acid, deoxynivalenol and its glucoside: In-house validation and application to wheat.

Nucleotide sugars, the activated forms of monosaccharides, are important metabolites involved in a multitude of cellular processes including glycosylation of xenobiotics. Especially in plants, UDP-glucose is one of the most prominent members among these nucleotide-sugars, as it is involved in the formation of glucose conjugates of xenobiotics, including mycotoxins, but also holds a central role in the interconversion of energized sugars such as the formation of UDP-glucuronic acid required for cell wall biosynthesis. Here, we present the first HILIC-LC-ESI-TQ-MS/MS method for the quantification of UDP-glucose and UDP-glucuronic acid together with the Fusarium toxin deoxynivalenol (DON) and its major plant detoxification product DON-3-O-glucoside (DON-3-Glc) utilizing a polymer-based column. For sample preparation a time-effective and straightforward 'dilute and shoot' protocol was applied. The chromatographic run time was minimized to 9min including proper column re-equilibration. In-house validation of the method verified its linear range, intra- (1-7%) and interday (8-20%) precision, instrumental LODs between 0.6 and 10ngmL(-1), selectivity and moderate matrix effects with mean recoveries of 85-103%. To prove the methods applicability, we analyzed two sets of wheat extracts obtained from different cultivars grown under standardized greenhouse conditions. The results clearly demonstrated the suitability of the developed method to quantify UDP-glucose, DON and its masked form D3G in diluted wheat extracts. We observed differing concentration levels of UDP-glucose in the two wheat cultivars showing different resistance to the severe plant disease Fusarium head blight. We propose that the higher ability to detoxify DON into DON-3-Glc might be a consequence of the higher cellular UDP-glucose pool in the resistant cultivar.

Isotope label-aided mass spectrometry reveals the influence of environmental factors on metabolism in single eggs of fruit fly.

In order to investigate the influence of light/dark cycle on the biosynthesis of metabolites during oogenesis, here we demonstrate a simple experimental protocol which combines in-vivo isotopic labeling of primary metabolites with mass spectrometric analysis of single eggs of fruit fly (Drosophila melanogaster). First, fruit flies were adapted to light/dark cycle using artificial white light. Second, female flies were incubated with an isotopically labeled sugar ((13)C(6)-glucose) for 12 h--either during the circadian day or the circadian night, at light or at dark. Third, eggs were obtained from the incubated female flies, and analyzed individually by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS): this yielded information about the extent of labeling with carbon-13. Since the incorporation of carbon-13 to uridine diphosphate glucose (UDP-glucose) in fruit fly eggs is very fast, the labeling of this metabolite was used as an indicator of the biosynthesis of metabolites flies/eggs during 12-h periods, which correspond to circadian day or circadian night. The results reveal that once the flies adapted to the 12-h-light/12-h-dark cycle, the incorporation of carbon-13 to UDP-glucose present in fruit fly eggs was not markedly altered by an acute perturbation to this cycle. This effect may be due to a relationship between biosynthesis of primary metabolites in developing eggs and an alteration to the intake of the labeled substrate - possibly related to the change of the feeding habit. Overall, the study shows the possibility of using MALDI-MS in conjunction with isotopic labeling of small metazoans to unravel the influence of environmental cues on primary metabolism.

The mechanism of galactosamine toxicity revisited; a metabonomic study.

1H NMR spectroscopy was used to investigate the metabolic effects of the hepatotoxin galactosamine (galN) and the mechanism by which glycine protects against such toxicity. Rats were acclimatized to a 0 or 5% glycine diet for 6 days and subsequently administered vehicle, galN (500 mg/kg), glycine (5% via the diet), or both galN and glycine. Urine was collected over 12 days prior to administration of galN and for 24 hours thereafter. Serum and liver tissue were sampled on termination, 24 hours post-dosing. The metabolic profiles of biofluids and tissues were determined using high-field 1H NMR spectroscopy. Orthogonal-projection to latent structures discriminant analysis (O-PLS-DA) was applied to model the spectral data and enabled the hepatic, urinary, and serum metabolites that discriminated between control and treated animals to be determined. Histopathological data and clinical chemistry measurements confirmed the protective effect of glycine. The level of N-acetylglucosamine (glcNAc) in the post-dose urine was found to correlate strongly with the degree of galN-induced liver damage, and the urinary level of glcNAc was not significantly elevated in rats treated with both galN and glycine. Treatment with glycine alone was found to significantly increase hepatic levels of uridine, UDP-glucose, and UDP-galactose, and in view of the known effects of galactosamine, this suggests that the protective role of glycine against galN toxicity might be mediated by changes in the uridine nucleotide pool rather than by preventing Kupffer cell activation. Thus, we present a novel hypothesis: that administration of glycine increases the hepatic uridine nucleotide pool which counteracts the galN-induced depletion of these pools and facilitates complete metabolism of galN. These novel data highlight the applicability of NMR-based metabonomics in elucidating multicompartmental metabolic consequences of toxicity and toxic salvage.

Nucleotides in sow colostrum and milk at different stages of lactation.

An experiment was conducted with the objective of measuring the concentrations of total milk solids (TMS), CP, and 5'monophosphate nucleotides in sow colostrum and milk. Twelve multiparous sows (Landrace x Yorkshire x Duroc) were used. Litter size was standardized at 11 piglets for all sows at farrowing. Sows were fed an 18.45% CP corn-soybean meal-based diet throughout lactation. The experimental period was the initial 28 d of lactation, with colostrum collected within 12 h of farrowing and milk collected on d 3, 7, 14, 21, and 28. Colostrum and milk samples were analyzed for TMS, CP, adenosine 5'monophosphate (5'AMP), cytidine 5'monophosphate (5'CMP), guanosine 5'monophosphate (5'GMP), inosine 5'monophosphate (5'IMP), and uridine 5'monophosphate (5'UMP). Total milk solids decreased (P < 0.05) from 26.7% on d 0 to 23.1% on d 3. The TMS further decreased (P < 0.05) to 19.3% on d 7, but remained relatively constant thereafter at 18.2, 18.8, and 19.2% on d 14, 21, and 28, respectively. The concentration of CP decreased from 16.6% in colostrum to 7.7, 6.2, 5.5, 5.7, and 6.3% in milk collected on d 3, 7, 14, 21, and 28, respectively (linear and quadratic effect; P < 0.05). Concentrations of 5'AMP, 5'CMP, 5'GMP, and 5'IMP increased from d 0 to d 3 and d 7, and then decreased during the remaining lactation period (quadratic effect; P < 0.05). The concentration of 5'UMP decreased from d 0 to 28 of lactation (linear and quadratic effects; P < 0.05). In colostrum, 5'UMP represented 98% of all 5'monophosphate nucleotides, and in milk, 5'UMP accounted for 86 to 90% of all nucleotides, regardless of day of lactation. The results of this experiment suggest that the concentrations of TMS and CP in sow mammary secretions changed during the first week of lactation, but were constant thereafter. Likewise, the concentrations of 5'monophosphate nucleotides changed during the initial week postpartum, but during the last 2 wk of a 4-wk lactation period, the concentrations were constant.

Quantification of uridine 5'-diphosphate (UDP)-glucose by high-performance liquid chromatography and its application to a nonradioactive assay for nucleoside diphosphate kinase using UDP-glucose pyrophosphorylase as a coupling enzyme.

We describe a method for the detection and quantification of nucleoside diphosphate kinase (NDPK). NDPK catalyzes the transfer of the gamma-phosphate of cytidine 5'-triphosphate on uridine 5'-diphosphate (UDP) to produce uridine 5'-triphosphate (UTP). The method uses a nonradioactive coupled enzyme assay in which UTP produced by NDPK is utilized by UDP-glucose pyrophosphorylase. This latter enzyme synthesizes UDP-glucose and inorganic phosphate in the presence of glucose 1-phosphate. UDP-glucose is detected at 260 nm after separation of the reaction mixture by high-performance liquid chromatography (HPLC) on a strong anion-exchange column. The assay is reliable, specific, and linear with respect to time and enzyme amount. Using 15 min incubation time, the method allows detection of NDPK activity below 10 pmol/min. It can be used to analyze kinetic behavior and to quantify NDPK from a wide variety of animal, microbial, and plant sources. It also provides an alternative to radiometric assays and an improvement on pyruvate kinase-linked spectrophotometric assays, which can be hampered by pigments present in crude extracts. Furthermore, we show that the HPLC method developed here can be directly used to assay enzymes for which UDP-glucose is a product.

Galactose metabolism in normal human lymphoblasts studied by (1)H, (13)C and (31)P NMR spectroscopy of extracts.

The development of tools to follow and quantitate the fate of galactose in mammalian cells is crucial to the study and understanding of the inherited disorders of galactose metabolism. In this study we incubated normal human lymphoblasts with 1- or 2-(13)C galactose for 2.5 or 5 h and prepared TCA extracts of the cells. The various galactose metabolites were identified and quantified using a combination of proton, carbon and phosphorus NMR spectra. Galactose-1-phosphate (gal-1P), uridine diphosphogalactose, uridine diphosphoglucose and galactitol were present in the extracts. Average levels for gal-1P were around 10 nmol/mg protein and for uridine diphosphoglucose, uridine diphosphogalactose and galactitol in the range of 0.5-2 nmol/mg protein. Galactonate was never found in any conditions. Percentage labeling could be estimated for gal-1P and for the ribose carbons of AMP. The labeling agrees with a conversion of galactose to glucose through the Leloir pathway.

Simultaneous, quantitative analysis of UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, UDP-glucose and UDP-galactose in human peripheral blood cells, muscle biopsies and cultured mesangial cells by capillary zone electrophoresis.

The aim of this study was to develop and evaluate a capillary zone electrophoretic (CE) procedure for the accurate quantification of the UDP-hexosamines as well as for the corresponding UDP-hexoses in samples from various biological origins. Testing different buffer conditions, voltages, capillary dimensions and temperatures, optimal results were achieved with a 90 mM borate buffer, pH 9.0, at 18 degrees C and 15.5 kV in an uncoated fused-silica capillary of 50 cm x 50 microm and a detection wavelength of lambda = 262 nm. The total procedure, i.e., including variations of the sample preparation, showed coefficients of variation for the peak areas between 4. 1% and 10.4% in mesangial cells (n = 7) and between 7.8 and 10.3% (n = 6) in leukocytes for the components of interest. To improve precision, an internal standard was used for calibration. The limit of detection for all compounds is an absolute amount of 180 fmol, sufficient for the precise analysis of UDP-sugars in a limited amount of biological samples, such as human leukocytes (obtained from a 10 mL blood sample), muscle biopsies (< or = 100 mg), and mesangial kidney cells (ca. 2.5 x 10(5) cells). This reproducible, quantitative analysis of all four UDP-sugars from various biomedically relevant origins by CZE is a definite improvement over the generally used high performance liquid chromatography (HPLC) procedures. The CZE method allows the study of the flux through the hexosamine pathway in diabetes mellitus and other diseases in a simple, quantitative and accurate way.

Advantages in the analysis of UDP-sugars by capillary electrophoresis-comparison of the conventional HPLC method with two new capillary electrophoretic micro-procedures.

Two different capillary electrophoretic separation modes for the analysis of UDP-sugars have been tested. In comparison to the traditionally used HPLC method the micellar electrokinetic capillary chromatography (MECC) as well as the capillary zone electrophoresis (CZE) results in an improvement of the separation profile and the speed of the analysis.

Acetaminophen glucuronidation accurately reflects gluconeogenesis in fasted dogs.

To assess whether acetaminophen glucuronide accurately reflects uridyl diphosphate-glucose (UDP-glucose) derived from gluconeogenesis during fasting, three mongrel dogs received infusions of [U-14C]lactate, [1-13C]galactose, and [6-3H]glucose (after fasting overnight or for 2.5 days). After initiation of the isotopes (3 h), acetaminophen was given, and the urinary acetaminophen glucuronide was isolated. The mean plasma [14C]glucose specific activity (SA) was similar to the mean urinary acetaminophen glucuronide SA both after fasting overnight [299 +/- 19 vs. 296 +/- 14 disintegrations.min-1 (dpm).mumol-1, respectively] and after 2.5 days of fasting (511 +/- 8 vs. 562 +/- 32 dpm/mumol, respectively). Mean plasma glucose flux calculated using [6-3H]glucose decreased (P < 0.05) with two additional days of fasting (18.7 +/- 1.2 vs. 13.6 +/- 0.6 mumol.kg-1.min-1), as did intrahepatic (P < 0.05) UDP-glucose flux measured using [1-13C]galactose (8.6 +/- 0.7 vs. 5.5 +/- 0.3 mumol.kg-1.min-1). We conclude that, in fasted dogs, plasma glucose and UDP-glucose, as sampled by acetaminophen, equally reflect gluconeogenesis and appear to come from the same pool of glucose 6-phosphate. In addition, cycling of glucose moieties through UDP-glucose and glycogen decreases with an increased period of fasting.

Sugar nucleotide concentrations in red blood cells of patients on protein- and lactose-limited diets: effect of galactose supplementation.

Uridine diphosphate (UDP) galactose, a pivotal compound in the metabolism of galactose, is the obligate donor of galactose in the formation of complex glycoconjugates. The cellular UDPgalactose concentration has been thought to be maintained by the interconversion of UDPglucose and UDPgalactose by UDPgalactose-4-epimerase. However, recent findings of lower average red blood cell (RBC) UDPgalactose concentrations in galactose-1-phosphate uridyltransferase-deficient patients suggest that other factors play a role in determining its concentration. To test the hypothesis that the amount of galactose traversing the Leloir pathway contributes to the cellular UDPgalactose pool, we determined RBC UDPgalactose in patients with maple syrup urine disease (MSUD), phenylketonuria (PKU), and other metabolic diseases who were treated with a low-protein, and consequently, low-lactose diet. Six patients with MSUD were also supplemented with 19 g galactose/d and their UDPhexose concentrations were measured at intervals. We show that young patients with MSUD or PKU have decreased average RBC UDPgalactose concentrations when compared with similarly aged healthy subjects. Galactose supplementation of MSUD patients significantly increased their UDPgalactose concentrations in both RBCs and white blood cells (WBCs) from 29.5 +/- 1.5 to 42.3 +/- 5.8 nmol/g hemoglobin and from 69.0 +/- 7.5 to 193.0 +/- 49.0 nmol/g protein, respectively. Discontinuation of supplementation was associated with a return to basal values in RBCs and a reattainment of the pretreatment ratio of UDPglucose to UDPgalactose in WBCs. These observations demonstrate that dietary galactose is a factor in establishing the steady state concentrations of the uridine sugar nucleotides and imply that galactose metabolism modulates the achievement of an epimerase-mediated equilibrium.

Detection and quantitation of phosphorus metabolites in crude tissue extracts by 1H and 31P NMR: use of gradient assisted 1H-31P HMQC experiments, with selective pulses, for the assignment of less abundant metabolites.

The analysis of crude tissue extracts by NMR has proven to be of use in the study of metabolism due to the non-destructive and non-selective character of the technique. Lists of 1H and 31P NMR assignments of phosphorus metabolites in water solution at specified pH and ionic composition are of large general value but their usefulness may be limited when analysing complex mixtures of metabolites at low concentrations. In this work we report on the use of gradient-assisted proton detected multiple quantum 1H and 31P coherence experiments with selective pulses for the rapid and unambiguous assignments of some crowded regions in 1H and 31P spectra of crude extracts from rat liver. The amplitudes of the gradient episodes were calibrated to optimize the coherence transfer pathway between proton and phosphorus, and the delay for the evolution of the long-range coupling was calculated from values of 3JPH and 4JPH ranging from 1.4 to 7.5 Hz. Moreover, a selective 90 degrees Gaussian pulse on the 31P channel was introduced to increase the resolution in the F1-domain and make the method even faster. The procedure was then applied to unambiguously assign the ID 31P and 1H spectra of perchloric acid extracts of rat livers that had been stimulated with phenylephrine, dBcAMP and glucagon and thus detect changes in the concentration of less abundant metabolites such as phosphoenolpyruvate, UDP-glucose and AMP. The fact that the quantification of these metabolites by either 31P and 1H methods lead to different results is discussed, and the use of 1H NMR spectroscopy for the quantification of phosphorus metabolites whose signal are too weak or poorly resolved in a 31P spectrum is proposed.

Cell wall polysaccharide biosynthesis and related metabolism in elicitor-stressed cells of French bean (Phaseolus vulgaris L.).

Enzyme activities involved in quantitative and qualitative flux of sugars into cell wall polysaccharides were determined following elicitor treatment of suspension cultured cells of French bean (Phaseolus vulgaris L.). Two subsets of activities were examined: the first were involved in synthesis and metabolism of UDP-glucose and the provision of the pool of UDP-sugars, and the second a selection of membrane-bound glycosyltransferases involved in the synthesis of pectins, hemicelluloses and glucans of the primary cell wall. Of the first group, only UDP-glucose dehydrogenase (EC 1.1.1.22) showed any significant induction in response to elicitor treatment, sucrose synthase (EC 2.4.1.13), UDP-glucuronate decarboxylase (EC 4.1.1.35), UDP-glucose and UDP-xylose 4-epimerases (EC 5.1.3.2 and EC 5.1.3.5 respectively) did not change in activity significantly over the time course. In contrast, enzymes of the second group showed a more complex response. Callose synthase (glucan synthase II, EC 2.4.1.12) increased in activity, as has been shown in other systems, while arabinan synthase (EC 2.4.1.-), xylan synthase (EC 2.4.1.72), xyloglucan synthase (EC 2.4.1.72) and glucan synthase I (EC 2.4.1.12) activities were rapidly depleted from membranes within 3 h following elicitor action. This rapid turnover of activity was striking, indicating that the half-life of such enzymes can be short and that elicitor action causes substantial perturbation of some membrane activities. Glucan synthase I activity appears to increase in the later stages over the time period measured, indicating some recovery of this metabolism.

13C-nuclear magnetic resonance spectroscopy studies of hepatic glucose metabolism in normal subjects and subjects with insulin-dependent diabetes mellitus.

To determine the effect of insulin-dependent diabetes mellitus (IDDM) on rates and pathways of hepatic glycogen synthesis, as well as flux through hepatic pyruvate dehydrogenase, we used 13C-nuclear magnetic resonance spectroscopy to monitor the peak intensity of the C1 resonance of the glucosyl units of hepatic glycogen, in combination with acetaminophen to sample the hepatic UDP-glucose pool and phenylacetate to sample the hepatic glutamine pool, during a hyperglycemic-hyperinsulinemic clamp using [1-13C]-glucose. Five subjects with poorly controlled IDDM and six age-weight-matched control subjects were clamped at a mean plasma glucose concentration of approximately 9 mM and mean plasma insulin concentrations approximately 400 pM for 5 h. Rates of hepatic glycogen synthesis were similar in both groups (approximately 0.43 +/- 0.09 mumol/ml liver min). However, flux through the indirect pathway of glycogen synthesis (3 carbon units-->-->glycogen) was increased by approximately 50% (P < 0.05), whereas the relative contribution of pyruvate oxidation to TCA cycle flux was decreased by approximately 30% (P < 0.05) in the IDDM subjects compared to the control subjects. These studies demonstrate that patients with poorly controlled insulin-dependent diabetes mellitus have augmented hepatic gluconeogenesis and relative decreased rates of hepatic pyruvate oxidation. These abnormalities are not immediately reversed by normalizing intraportal concentrations of glucose, insulin, and glucagon and may contribute to postprandial hyperglycemia.

Separation and analysis of 4'-epimeric UDP-sugars by ion-pair reversed-phase HPLC.

A simple and sensitive method for determination of 4'-epimeric UDP-sugars using ion-pair reversed-phase HPLC has been developed. The method presents advantages over existing ion-exchange HPLC procedures mainly concerning sensitivity and rapidity of analysis as well as efficiency and stability of the column. It is based on the ability of borate ions to react with cis-diols resulting in the formation of UDP-sugar-borate complexes with different charges. Good resolution and rapid separation (5-25 min) of all 4'-epimeric UDP-sugars tested was achieved with this method, was suitable for concentrations over 20 pmol. The applicability to biochemical analysis was demonstrated by the quantitative determination of the UDP-2-deoxyglucose and UDP-2-deoxygalactose formed in yeast cells upon incubation in the presence of 2-deoxygalactose.

A role for glycosphingolipid accumulation in the renal hypertrophy of streptozotocin-induced diabetes mellitus.

Glucosylceramide (GlcCer) and related glycosphingolipids have been implicated as causal elements in both the growth of cells and in the regulation of hormonal signaling. We therefore studied whether the renal hypertrophy induced by diabetes was associated with enhanced synthesis of glycosphingolipids. 16 d after the induction of diabetes, increases in renal size and concentration of glucocerebroside and ganglioside GM3 were observed paralleling an increase in UDP-Glc concentration. GlcCer synthase and beta-glucosidase-specific activities were no different between control and diabetic kidneys. The apparent Km of the GlcCer synthase with respect to UDP-Glc was 250 microM and was unchanged in the diabetic kidneys. The observed concentrations of UDP-Glc were 149 and 237 microM in control and diabetic kidneys, respectively. The UDP-Glc level is thus rate limiting with regard to GlcCer synthesis. To determine whether the changes in glycolipid content were functionally significant, diabetic and control groups were treated with the GlcCer synthase inhibitor, D-threo-1-phenyl-2-decanoyl-amino-3-morpholino-1- propanol, 2 wk after the induction of diabetes. Kidney weights in the diabetic rats treated with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol were no different than the control groups. Morphometric analysis of glomerular volumes paralleled changes in renal growth. Glycosphingolipid formation may therefore represent a significant pathway for glucose utilization in early diabetic nephropathy.